Rabbit muscle glycogen debranching enzyme (amylo-1- 6-glucosidase/4-Alpha-glucanotransferase) is the only multicatalytic site mammalian (eucaryotic) enzyme to be reported that is active as a monomer. The enzyme, a single polypeptide molecule with a molecular weight of 160-170,000, consists of two separate enzymatic activities which have different modes of action. The debranching enzyme exists along with phosphorylase and its activating enzyme system as a component of a protein-glycogen multi-enzyme eomplex in muscle which is involved with glycogen metabolism and linked to muscle contractions. Investigation of the relationship between the two debrancher activities has led to a mechanism of how the enzyme functions in debranching and given some insight into how its active site is constructed: there is a separate glucosidase site, transferase site, and polymer binding site. The debrancher forms an active glucosyl intermediate complex at the glucosidase site that can be isolated. Upon denaturation the intermediate forms a covalently linked glucosyl-enzyme adduct which can be used to label the site. A covalently linked maltotriosyl adduct can also be formed at the transferase site. The present study involves a continuation of an investigation of the debrancher as a protein molecule to determine how the two activities (glucosidase and transferase) relate to each other as structural components of the polypeptide and a study of their catalytic mechanisms. The covalently linked glycosyl adducts will be used to locate the glucosidase and transferase domains on the polypeptide. The anomeric configuration of the glucosyl-enzyme linkage will be determined and the chemical reactivity of the active intermediate will be studied. Similar studies will be conducted with the transferase intermediate. The kinetic properties of the transferase only form of the enzyme will be studied and asymmetric limit dextrin prepared using it. An effort will be made to prepare large crystals of the debrancher for X-ray crystallography structural studies. This study will delineate the relationship between the structure nd function of the debrancher. It will also give further insight into its relationship with phosphorylase as a component of the glycogenolytic complex.